Delineation of the molecular mechanisms of directed motility is important for understanding the processes of cancer metastasis and invasion. The processes underlying the migration of primary tumor cells to ectopic sites involve the reorganization of the actin cytoskeleton. Several factors that regulate changes in the actin cytoskeleton of motile cells have been identified, however, the signaling molecules that direct the de novo polymerization of actin and its ultimate spatial and dynamic organization need to be clarified. Several members of the Rho family of small GTPases (Rho, Rac and Cdc42) have been identified as key signal transducers that link cell surface receptors to the actin cytoskeleton. We aim to test the hypothesis that Rac and Cdc42 are important regulators of breast cancer cell metastasis. Our research plan involves general strategy of using molecular genetics approach in combination with cellular and biochemical techniques. The construction of stable transfectants of human and rat mammary carcinoma cells with dominant active, dominant negative, and effector domain mutations of Rac and Cdc42 will be performed. We will evaluate the effect of activating or inactivating Rac and Cdc42 on breast cancer metastasis. These transfectants will be characterized by cellular, biochemical, and histopathological analysis. The metastatic potential of the transfected breast cancer cells expressing mutant Rac and Cdc42 will be assessed following injection into rats (rat mammary carcinomas) and nude mice (human mammary carcinomas). This research is expected to establish Rac and/or Cdc42 as important mediators of cancer metastasis. Our long-term goal is to delineate the signaling cascades mediated by Rac and/or Cdc42 that underlie invasion and metastasis. Identifying such key regulators of directed migration is expected to increase knowledge of the cellular and molecular basis of metastasis and may also lead to novel approaches in cancer therapy.